Hu Jiandong, Wang Tingting, Wang Shun, Chen Mingwen, Wang Manping, Mu Linying, Chen Hongyin, Hu Xinran, Liang Hao, Zhu Juanhua, Jiang Min
Department of Electrical Engineering, Henan Agricultural University, Zhengzhou, China; State key laboratory of wheat and maize crop science, Zhengzhou, China.
Department of Electrical Engineering, Henan Agricultural University, Zhengzhou, China.
PLoS One. 2014 Oct 29;9(10):e111292. doi: 10.1371/journal.pone.0111292. eCollection 2014.
A sensitive and label-free analytical approach for the detection of porcine circovirus type 2 (PCV2) instead of PCV2 antibody in serum sample was systematically investigated in this research based on surface plasmon resonance (SPR) with an establishment of special molecular identification membrane. The experimental device for constructing the biosensing analyzer is composed of an integrated biosensor, a home-made microfluidic module, and an electrical control circuit incorporated with a photoelectric converter. In order to detect the PCV2 using the surface plasmon resonance immunoassay, the mercaptopropionic acid has been used to bind the Au film in advance through the known form of the strong S-Au covalent bonds formed by the chemical radical of the mercaptopropionic acid and the Au film. PCV2 antibodies were bonded with the mercaptopropionic acid by covalent -CO-NH- amide bonding. For the purpose of evaluating the performance of this approach, the known concentrations of PCV2 Cap protein of 10 µg/mL, 7.5 µg/mL, 5 µg/mL, 2.5 µg/mL, 1 µg/mL, and 0.5 µg/mL were prepared by diluting with PBS successively and then the delta response units (ΔRUs) were measured individually. Using the data collected from the linear CCD array, the ΔRUs gave a linear response over a wide concentration range of standard known concentrations of PCV2 Cap protein with the R-Squared value of 0.99625. The theoretical limit of detection was calculated to be 0.04 µg/mL for the surface plasmon resonance biosensing approach. Correspondingly, the recovery rate ranged from 81.0% to 89.3% was obtained. In contrast to the PCV2 detection kits, this surface plasmon resonance biosensing system was validated through linearity, precision and recovery, which demonstrated that the surface plasmon resonance immunoassay is reliable and robust. It was concluded that the detection method which is associated with biomembrane properties is expected to contribute much to determine the PCV2 in sample solutions instead of PCV2 antibody in serum samples quantitatively.
本研究基于表面等离子体共振（SPR），通过建立特殊的分子识别膜，系统地研究了一种灵敏且无标记的分析方法，用于检测血清样本中的猪圆环病毒2型（PCV2）而非PCV2抗体。构建生物传感分析仪的实验装置由集成生物传感器、自制微流控模块以及包含光电转换器的电控电路组成。为了利用表面等离子体共振免疫分析法检测PCV2，巯基丙酸已预先通过巯基丙酸的化学基团与金膜形成的强S - Au共价键的已知形式与金膜结合。PCV2抗体通过共价 -CO-NH-酰胺键与巯基丙酸结合。为了评估该方法的性能，通过依次用PBS稀释制备了已知浓度为10 µg/mL、7.5 µg/mL、5 µg/mL、2.5 µg/mL、1 µg/mL和0.5 µg/mL的PCV2 Cap蛋白，然后分别测量其增量响应单位（ΔRUs）。利用从线性CCD阵列收集的数据，在PCV2 Cap蛋白标准已知浓度的宽浓度范围内，ΔRUs给出了线性响应，R平方值为0.99625。表面等离子体共振生物传感方法的理论检测限计算为0.04 µg/mL。相应地，获得了81.0%至89.3%的回收率。与PCV2检测试剂盒相比，该表面等离子体共振生物传感系统通过线性、精密度和回收率进行了验证，表明表面等离子体共振免疫分析法可靠且稳健。得出的结论是，与生物膜特性相关的检测方法有望在定量测定样品溶液中的PCV2而非血清样品中的PCV2抗体方面做出很大贡献。
